Medicament for treatment of liver cancer

ABSTRACT

The invention provides a pharmaceutical composition comprising Sorafenib in combination with an inhibitor of a specific kinase inhibitor as a medicament for the treatment or prevention of liver cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 14/131,059, filed Apr. 10, 2014, which is a 371 national phaseof PCT/EP2012/063445, filed Jul. 9, 2012, which claims the benefit ofthe filing date of U.S. Provisional Application No. 61/508,368, filedJul. 15, 2011, which claims priority to European Application No.11173379.6, filed Jul. 8, 2011, European Application No. 12162905.9,filed Apr. 2, 2012 and European Application No. 12161141.2, filed Mar.23, 2013, the disclosures of which are incorporated, in their entirety,by this reference.

The present invention relates to a medicament and to a pharmaceuticalcomposition, respectively, which is suitable for the treatment orprevention of liver cancer, especially hepatocellular carcinoma (HCC) inhuman patients.

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .txt format. The .txtfile contains a sequence listing entitled “16718_3_Seq_Listing”previously filed in the parent application Ser. No. 14/131,059, filedApr. 10, 2014, and saved on Aug. 30, 2019 and is 510.46 megabytes insize. The sequence listing contained in this .txt file is part of thespecification and hereby incorporated by reference in its entirety.

Further, the invention relates to a method for the treatment of livercancer by administration of the pharmaceutical composition to a patient,and to a process for producing a medicament or pharmaceuticalcomposition, respectively, which is suitable for the treatment of livercancer.

STATE OF THE ART

Today, the only curative options for the treatment of liver cancer aresurgical resection or liver transplantation. However, at the time ofdiagnosis the majority of patients present with advanced tumor growthand are therefore not eligible for these treatment options. Liver canceris a primarily chemoresistant tumor and only recently Llovet andcoworkers described the multikinase inhibitor Sorafenib as the firstsystemic treatment which can prolong survival of patients with HCC.However, the treatment is costly and only yields a survival advantage ofless than three month. Therefore, there is an urgent clinical need forthe development of medicaments which increase efficiency over Sorafenib.

WO 2010/128259 A1 describes a pharmaceutical combination of Sorfenibwith a vascular disrupting agent.

WO2005/009961 A2 describes a fluoro derivative of Sorafenib.

Huynh et al, Biochemical Pharmacology, 550-560 (2010), describeSorafenib for use as a medicament in the treatment of liver cancer.

Noel et al, Med Oncol 323-330 (2008) report on studies on cancertreatment using Sorafenib or Thalidomid (Contergan).

Parekh et al, Experimental and Toxicologic Pathology 167-173 (2011)describe that resveratrol treatment downregulated cyclin D1 and p38 MAPkinase, Akt and Pak1 expression and activity in HepG2 cells, and thatresveratrol treated cells showed an increase in ERK activity.

Min et al, Seminars in Cancer Biology, 10-20 (2011), quote that p38α hasa negative regulatory role in tumorigenesis, especially of the liver,and that the p38α signalling pathway has a suppressive role intumorigenesis. Sorafenib is described to inhibit proliferation of HCCcells and to induce apoptosis by inhibiting the phosphorylation of MEK,ERK and down-regulating cyclin D1 levels.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a pharmaceutical compositionfor the treatment of liver cancer with an increased efficacy. It is apreferred object of the invention to provide for a pharmaceuticalcomposition for use against liver cancer, the composition having anincreased efficacy in comparison to a composition containing the sameamount of Sorafenib.

GENERAL DESCRIPTION OF THE INVENTION

The invention achieves these objects by the features as defined by theclaims, especially by providing a pharmaceutical composition, thecomposition comprising Sorafenib and, in addition to Sorafenib, aninhibitor of Mapk14 (p38a kinase inhibitor) as a medicament for use inthe treatment and/or in the prevention of cancer, especially livercancer. The inhibitor of Mapk14 activity is a pharmaceutical activeagent which in combination with Sorafenib is used as a medicament forthe treatment and/or prevention of liver cancer. In the combination,Sorafenib and the inhibitor of Mapk14 can be provided for jointadministration, e.g. as a composition containing both Sorafenib and aninhibitor of Mapk14, or Sorafenib and the inhibitor of Mapk14 can beprovided for separate administration, e.g. firstly Sorafenib andsecondly an inhibitor of Mapk14, or firstly an inhibitor of Mapk14 andsecondly Sorafenib. Accordingly, the invention provides a combination ofSorafenib with an inhibitor of Mapk14 as a medicament for the treatmentand/or prevention of liver cancer for joint or separate administration,as well as for a composition comprising a combination of Sorafenib andof an inhibitor of Mapk14, e.g. each of Sorafenib and an inhibitor ofthe activity of Mapk14 contained in a separate formulation for use inseparate administration, or a formulation containing both Sorafenib andan inhibitor of the activity of Mapk14 in combination. Further, theinvention relates to the treatment of cancer, especially of livercancer, by administration of this pharmaceutical composition, e.g byseparate or simultaneous administration of Sorafenib and of theinhibitor of Mapk14 to a human suffering from cancer, especially fromliver cancer. Optionally, the liver cancer is non-viral liver cancer orvirally caused liver cancer.

It has been demonstrated during the preparation of the invention that anagent which inhibits the activity of Mapk14 increases the efficacy ofSorafenib, as a medicament in the treatment or prevention of livercancer. As the inhibition of the activity of Mapk14 can be obtained bythe inhibition of the expression of Mapk14 gene product or protein,inhibitors of the translation of the mRNA encoding Mapk14, andinhibitors of Mapk14 protein activity, e.g. kinase inhibitors, arecomprised in the group of inhibitors of the activity of Mapk14 which areused in a combination with Sorafenib for use as a medicament for thetreatment or prevention of liver cancer.

Inhibitors of the activity of Mapk14, especially of the expression ofMapk14, are comprised in the group of small regulatory RNAs such assmall inhibitory RNAs (siRNA), short hairpin RNA (shRNA) and microRNAs(miRNA), having a nucleic acid sequence hybridizing under physiologicalconditions, e.g. within a liver cell, to the mRNA encoding Mapk14 toreduce or inhibit the presence of Mapk14 in a hepatocyte or a livercancer cell through RNA interference and accordingly inhibit theactivity of Mapk14. Small regulatory RNA molecules comprise or consistof the group containing at least one of the following inhibitory RNAs:SEQ ID NO: 1 to SEQ ID NO: 1364.

ShRNA molecules, e.g. contained in microRNA molecules, hybridize to themRNA encoding Mapk14. The Mapk14 encoding gene gives rise to four mRNAs,which are regarded as splice products. The splice product mRNAs aregiven as SEQ ID NO: 1365 to SEQ ID NO: 1368. The shRNA of the inventionthrough RNA interference reduce the expression, and hence the activityof Mapk14 gene product. Alternatively, the inhibitory RNAs can becontained in classical antisense oligonucleotides for targeting the samemRNA regions for suppression of Mapk14 expression.

The specificity of shRNA, especially when the sequence encoding theshRNA was contained in a microRNA can also be shown in an vitro testusing the reduction of the expression of a reporter gene product from afusion gene, which produces a fusion mRNA that contains the codingsequence for both the reporter gene and for Mapk14. In this assay, itcan be found that the mRNA of the fusion gene, which mRNA comprises thecoding sequence for Mapk14 in combination with the coding sequence forthe reporter gene, is reduced in the presence of an shRNA which isspecific for the mRNA encoding Mapk14, whereas a control encoding anmRNA of the reporter gene only did not show a reduction of the reportergene expression in the presence of an shRNA hybridizing to the mRNAencoding Mapk14.

The inactivation of Mapk14 by inhibiting or reducing the expression ofMapk14 in hepatocytes or liver cancer cells in experimental animals,both by suppression of the expression of Mapk14 via continuousexpression of an shRNA specific for the mRNA encoding Mapk14 in themouse model, and by administration of a pharmaceutical composition, inwhich the pharmaceutical active agent consists of an shRNA specific forthe mRNA encoding Mapk14 of the mouse reduces the present liver canceror prevents the generation of liver cancer upon administration of agentswhich in the absence of the combination of a Mapk14 inhibitor andSorafenib would induce a liver cancer.

The reduction or repression of Mapk14 activity by reducing orsuppressing the expression of Mapk14 using the continuous expression ofan shRNA which is specifically directed against the mRNA encodingMapk14, or by administration of a pharmaceutical composition containingas the active ingredient or agent an shRNA specifically directed againstthe mRNA encoding Mapk14 in the animal model reduces a previouslyestablished liver cancer with increased efficacy upon administration ofSorafenib in comparison to the treatment with Sorafenib as the onlypharmaceutical active agent, i.e. Sorafenib without the combination withan agent reducing Mapk14 activity.

In a preferred embodiment, the inhibitor of the activity of Mapk14 is atleast one of the group comprising or consisting of inhibitors which havepreference or specificity for inactivating the Mapk14 gene product,which compounds are given in the following table 1. Generally, aninhibitor of Mapk14 according to the invention has an IC50 of at maximum30 nM, preferably of at maximum 20 nM, more preferably of at maximum 10nM or max. 5 nM or of at maximum 1 nM, most preferably of at maximum 0.1or of at maximum 0.01 nM. Preferably, the IC50 is determined in an invitro assay on Mapk14, e.g. determining the IC50 (inhibitoryconcentration for 50% inhibition) in dependence on the concentration ofthe inhibitor. For example, Mapk14, preferably having the human aminoacid sequence and human structure, e.g. produced by expression of thehuman gene encoding Mapk14 in a human cell line, is isolated andincubated in a suitable buffer in the presence of a substrate to bephosphorylated, e.g. ATF-2, MAPKAPK-2 or Hsp27, ³²P-γ-labelled ATP andthe inhibitor. The ³²P-γ-labelled ATP is separated from the substrate,and the amount of ³²P-phosphorylation is determined, e.g. usingscintillation counting. The preferred substrate are ATF-2, MAPKAPK-2 orHsp27 and a preferred buffer is: 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 1mg/ml BSA and protease inhibitor e.g. Complete mini (Roche), usingincubation at room temperature or 37° C. For comparison, the inhibitorof Mapk14 has a lower IC50 than Sorafenib, which has an IC50 for Mapk14of approx. 38 nM.

Additionally or alternatively, the inhibitory activity of the inhibitorof Mapk14 is determined in a cell-based in vitro assay using e.g. cancercells, e.g. Hep3B. In the assay, the inhibitory activity of p38inhibitors is determined for substrate protein of Mapk14 in relation toa housekeeping gene, e.g. α-tubulin. In the assay, cells are incubatedwith various concentrations of the inhibitor or carrier onlyrespectively, e.g. for 2-4 days, and protein is extracted, e.g. usingNP40-containing buffer. In the extracted protein, phosphorylatedsubstrate protein of Mapk14 is determined, e.g. by immunologicaldetection of phosphorylated substrate protein, e.g. using a Western blotof SDS-PAGE separated cellular protein. For immunological detection, aphospho-specific antibody can be used. A preferred substrate protein isATF-2, MAPKAPK-2 and/or Hsp27.

Additionally or alternatively, the combination of Sorafenib with theadditional inhibitor of Mapk14 of the invention in a cell-based assayhas a significant higher toxicity or inhibition of proliferation rateagainst cultivated cancer cells, especially against liver cancer cells,e.g. against Hep3B. In the assay, cultivated living cells are countedfollowing incubation with a combination of Sorafenib and the inhibitorof Mapk14 or with the same concentration of Sorafenib or inhibitoragainst Mapk14 alone or carriers, respectively, e.g. at concentrationsof at maximum 20 μM, preferably at maximum 12 μm, more preferably atmax. 5 μm. Significance can be determined using the two-tailed student'sT-test. Preferably, the combination of Sorafenib and the inhibitor ofMapk14 according to the invention reduce cell proliferation of has aninhibitory effect on the cultivated cancer cells higher by at least afactor of 2, preferably at least by a factor of 4, more preferably atleast by a factor of 10, in comparison to Sorafenib of the sameconcentration by itself.

Unless otherwise defined, substituents R1 to R14 can be selectedindependently from the group comprising CH₃, CH₂CH₃, CH₂CH₃CH₃,CH₂(CH₂)₂CH₃, CH₂══CH₂, H, F, Cl, Br, I, SO₂, CF₃, N₃, OH, NH₂, ═O,N(CH₃)₂, OMe, OEt, SO₂Me.

In Table 1, IC50 values are for Mapk14.

In the above Table, references to tradenames, trivial names andespecially to publication number of patents or patent applicationsinclude the subject-matter of such references and publications into thepresent application, especially in respect of characterizations andmethods for synthesis of the compounds.

It was found in in vitro assays using cultivated liver cancer cells ofboth murine and human origin, respectively, that the efficacy ofSorafenib against liver cancer cells was increased in the presence of aninhibitor of Mapk14 activity. As examples for inhibitors of Mapk14activity, BIRB-796, SB 202190, or SX 011 were used, which are kinaseinhibitors that act upon and/or are specific for Mapk14 protein.

DETAILED DESCRIPTION OF THE INVENTION

The invention is now described by way of examples with reference to thefigures, wherein

FIG. 1 schematically shows the constructs used for geneticallymanipulating mice for generation of liver carcinomas with concurrentexpression of shRNAs which can be non-specific or specific for the mRNAencoding Mapk14,

FIG. 2 shows the survival rates of mice expressing different shRNAmolecules with and without administration of Sorafenib,

FIG. 3 shows the detected levels of mRNA specific for Mapk14 withexpression of shRNAs which are non-specific or specific for the mRNAencoding Mapk14,

FIG. 4 shows a Western blot with specific detection for Mapk14 proteinlevels from cells expressing shRNA with or without specificity forMapk14 mRNA, and with detection for α-tubulin as a loading control,

FIG. 5 shows micrographs and GFP images of explanted mouse livers withand without administration of Sorafenib and expression of shRNAsspecific for Mapk14 mRNA and controls,

FIG. 6 schematically depicts the mechanism of the doxycycline dependenttranscription of shRNAs and the GFP marker gene,

FIG. 7 shows the survival rates of mice treated with Sorafenib or acontrol compound (carrier) in the presence of the shRNA specific for themRNA encoding Mapk14, (which transcription was activated 5 days aftertumor development) and in the absence of the shRNA specific for the mRNAencoding Mapk14, as well as control shRNAs,

FIG. 8 shows the number of cells (as an indicator of proliferativeactivity) in an in vitro assay in which murine liver cancer cells weretreated with Sorafenib in combination with the Mapk14 inhibitorBIRB-796,

FIG. 9 shows the number of cells (as an indicator of proliferativeactivity) in an in vitro assay, in which a human liver cancer cell linewas treated with Sorafenib in combination with the inhibitor of Mapk14BIRB-796,

FIG. 10 shows a picture of cell staining (cell density as an indicatorof proliferative activity) (crystal violet assay) of a human livercancer cell line after treatment of Sorafenib in combination with theMapk14-specific inhibitor BIRB-796,

FIG. 11 shows the quantification of the number of dead cells (humanliver cancer cell line) after treatment with Sorafenib in combinationwith the Mapk14-specific inhibitor BIRB-796 and controls, respectively,

FIG. 12 shows the number of cells in an in vitro assay in which a murinehepatocyte cancer cell line was treated with Sorafenib in combinationwith the Mapk14 inhibitor SB202190,

FIG. 13 shows the number of cells in an in vitro assay, in which a humanliver cancer cell line was treated with Sorafenib in combination withthe inhibitor of Mapk14 SB202190,

FIG. 14 shows a picture of cell staining (cell density as a marker forproliferative activity) of a human liver cancer cell line aftertreatment of Sorafenib in combination with the Mapk14-specific inhibitorSB202190,

FIG. 15 shows the quantification of the number of dead cells (humanliver cancer cell line) after treatment with Sorafenib in combinationwith the Mapk14-specific inhibitor SB202190 and controls, respectively,

FIG. 16 shows the number of cells in an in vitro assay in which a murineliver cancer cell line was treated with Sorafenib in combination withthe Mapk14 inhibitor SX011, and

FIG. 17 shows the the number of cells in an in vitro assay, in which ahuman liver cancer cell line was treated with Sorafenib in combinationwith the inhibitor of Mapk14 SX011.

EXAMPLE 1 Treatment of Liver Cancer In Vivo

As a representative of a human patient having liver cancer, agenetically manipulated mouse model (p19^(Arf−/−)) was generated, inwhich liver cancers were induced by a constitutive expression of theoncogenic NrasG12V mutant.

The nucleic acid construct for generating liver carcinomas isschematically shown in FIG. 1, containing an expression cassette,wherein the coding sequence for NrasG12V is arranged between a 5′promoter sequence and a 3′ polyadenylation site, which expressioncassette is flanked by two inverted repeat elements (IR). Forintrahepatic delivery of the expression cassette, this nucleic acidconstruct was administered in combination with a nucleic acid constructcontaining an expression cassette for the transposase sleeping beauty 13(SB13) under the control of the constitutive phosphoglycerate kinasepromoter (PGK). The mouse model receiving both nucleic acid constructsshown in FIG. 1 was p19^(Arf−/−), providing the genetic backgroundsufficient for constitutive generation of murine liver cancer. Nucleicacid constructs were introduced into experimental mice usinghydrodynamic tail vein injection.

Mice that were genetically conditioned to develop liver cancer weretreated with a pharmaceutical composition containing Sorafenib in acarrier, and carrier alone as a control.

The expression cassette in addition to the coding sequence for Nras12Vcontains the coding sequence for a short hairpin RNA as an example foran siRNA/shRNA. As an shRNA, a non-specific shRNA (shcontrol) was used,an shRNA specific for the murine mRNA encoding murine Mapk14(shMapk14.1095), and an alternative shRNA specific for the murine mRNAencoding Mapk14 (shMapk14.2590). For each mouse model having one of thenucleic acid constructs, mice were mock-treated with carrier, or withSorafenib alone.

The survival curves are shown in FIG. 2. A liver cancer patient withouta Mapk14 inhibitor is represented by the non-specific shRNA (1,shcontrol+carrier) and with the known treatment of Sorafenib (2,shcontrol+Sorafenib) according to the mouse model has an increasedsurvival rate, whereas both mouse models in which the Mapk14 activity isinhibited by the shRNA specific for the mRNA encoding Mapk14 incombination with Sorafenib (4, shMapk14.1095+Sorafenib, and 6,shMapk14.2590+Sorafenib) have a significantly increased survival rate.In contrast, the inactivation of Mapk14 alone by an shRNA, i.e. withouttreatment by administration of Sorafenib (3, shMapk14.1095+carrier and5, shMapk14.2590+carrier) have a survival rate essentially correspondingto the survival rate of mouse models without inactivation of Mapk14 (1,3, 5).

FIG. 3 shows the levels of mRNA encoding Mapk14 in relation (%) to thenon-specific control shRNA (shcontrol). It can be seen that theexpression of each of shMapk14.1095 (SEQ ID NO: 1370) and shMapk14.2590(SEQ ID NO: 1371) and of shMapk14.2364, which hybridize to the mRNAencoding Mapk14 results in a reduction of the mRNA encoding Mapk14 inthe liver tissue.

FIG. 4 shows Western blot analyses of murine liver cancer cells withspecific detection of Mapk14 and α-tubulin as a loading control. Thisanalysis shows that the expression level

of Mapk14 is significantly reduced by expression of shRNA specific forthe mRNA encoding Mapk14, exemplified by shMapk14.1095, shMapk14.2590,and shMapk14.2364, whereas there is a drastically higher level of Mapk14expression in the presence of shRNA (shcontrol) expression.

FIG. 5 shows photographs of livers explanted from the mouse model andthe respective GFP—imaging of explanted mouse livers, confirmingvisually that in mouse models receiving no Sorafenib (top row, FIGS.5.1, 5.2, 5.5, 5.6, 5.9, and 5.10) (carrier), the expression of anon-specific shRNA (shcontrol, FIGS. 5.1, 5.2) or of an shRNAinactivating Mapk14 (shMapk14.1095, shMapk14.2590, FIGS. 5.5, 5.6, 5.9,and 5.10) essentially do not reduce the development of liver cancer.

In a further experiment it was observed that in further control micewhich did not express an shRNA, a similar tumor development and asimilar survival rate was found as for the mice expressing thenon-specific shRNA (shcontrol).

The lower row of pictures of FIG. 5 (FIGS. 5.3, 5.4, 5.7, 5.8, 5.11, and5.12) shows that the inactivation of Mapk14, in this assay obtained invivo by expression of an shRNA specific for the mRNA encoding Mapk14(5.7, 5.8, 5.11, and 5.12) in the presence of treatment with Sorafenibdrastically reduces the occurrence of liver cancer both in relation tothe treatment with Sorafenib alone (FIGS. 5.3 and 5.4) and in relationto expression of an inhibitory shRNA alone (FIGS. 5.5, 5.6, 5.9, and5.10).

The GFP imaging correlates with expression of NrasG12V positive tumors.Decreased GFP activity is observed for shRNAs shMapk14.1095 andshMapk14.2590 in the presence of Sorafenib, indicating reduction ofcancer cells that were induced by the expression of Nras.

EXAMPLE 2 Treatment of Liver Cancer

Again, mouse models were used for representing human liver cancerpatients in treatment using Sorafenib in combination with an agentinhibiting the activity of Mapk14. As an example for a pharmaceuticalagent for the inactivation of the activity of Mapk14, an shRNA(shMapk14.1095) specific for the murine mRNA encoding Mapk14 was used.The shRNA was provided by controlled expression using an expressioncassette which is under the control of a tetracycline response element(TRE) of the otherwise constitutive viral promoter (P_(minCMV)). Theexpression cassette contains the coding sequence for a shRNA moleculeand the coding sequence of a GFP marker gene. The nucleic acid constructcontaining the expression cassette for the shRNA molecule and the codingsequence of a GFP marker gene under the control of the Tet-induciblepromoter also contains a constitutive expression cassette for rtTA(rtetRVP16), which in the presence of Doxycycline (DOX) binds to the TREand activates the promoter of the expression cassette encoding a fusionof shRNA and GFP.

FIG. 6 schematically shows the nucleic acid constructs and the mechanismfor inducing transcription of shRNA and GFP encoding sequence.

Generally, the nucleic acid construct of FIG. 6 is introduced intoexperimental mice via retroviral infection of cancer cells which afterselection are injected into the liver of wildtype mice. The injectedmice then develop liver carcinomas in which Mapk14 can be conditionallyinactivated by inducing transcription of the shRNA by addition of DOX.

FIG. 7 shows the result of an experiment using the expression of anshRNA specific for the mRNA encoding murine Mapk14, namelyshMapk14.1095, or a non-specific shRNA (shcontrol). The administrationof DOX induces inactivation of Mapk14 by inducing transcription of theshRNA and GFP. The administration of DOX in combination with Sorafenibsignificantly increased survival rates (10,shMapk14.1095+DOX+Sorafenib), whereas in the absence of the inactivationof Mapk14, represented by the lack of induction of shMapk14 in theabsence of DOX (14, shMapk14.1095−DOX+Sorafenib) or in the presence of anon-specific shRNA (12, shcontrol+DOX+Sorafenib) showed a lower survivalrate as expected for treatment by Sorafenib alone. For control, theabsence of a specific inhibitory shRNA and without Sorafenib (15,shMapk14.1095−DOX−Sorafenib), in the absence of non-specific shRNA andwithout Sorafenib (13, shcontrol−DOX−Sorafenib), and with presence ofMapk14 specific shRNA without Sorafenib but carrier (11,shMapk14.1095+DOX+carrier) were tested.

EXAMPLE 3 Inactivation of Mapk14 by a Specific Kinase InhibitorIncreases the Efficacy of Sorafenib Against Liver Cancer Cell Lines

As an example for a medicament containing Sorafenib in combination withan inhibitor specific for Mapk14 kinase, BIRB-796, SB 202190 and SX 001were tested in an in vitro assay. These in vitro experiments show theincreased efficacy of Sorafenib when administered in combination with aninhibitor of Mapk14 kinase protein on the example of cultivated murineand human liver cancer cell lines.

In the assay, murine liver cancer cells (Nras arf−/−) were cultivated.Cultivated cells were treated with 10 μM Sorafenib, 2 μM BIRB, 12 μMBIRB and with a combination of 10 μM Sorafenib+2 μM BIRB, or acombination of 10 μM Sorafenib+12 μM BIRB. For each of these assays, aparallel assay was made, replacing Sorafenib or Mapk14 inhibitors byformulation agents without pharmaceutical active agent (carrier).

FIG. 8 shows the results of the viable cell count after two days oftreatment of the murine cancer cells and FIG. 9 shows the viable cellcount after 7 days of treatment of the cultivated human liver cancercell line (Hep3B) with the combinations of 2 μM Sorafenib, 2 μM BIRB, 12μM BIRB, a combination of 2 μM Sorafenib+2 μM BIRB, or a combination of2 μM Sorafenib+12 μM BIRB, respectively, in the left columns, whereassamples without pharmaceutical active agent (carrier) are shown as righthand columns. These results show that the activity of Sorafenib againstliver cancer cells can be reproduced in vitro with cultivated murine andhuman liver cancer cells, respectively. In detail, the presence of 10 μMor 2 μM Sorafenib as the only pharmaceutical active agent shows adecrease of viable cancer cells after two and seven days, respectively,whereas in murine cells, the inhibitor BIRB-796 only has no significantinfluence on cultivated murine liver cancer cells, whereas BIRB-796alone shows a reduction of viable human cancer cells. In murine livercancer cells, and even more pronounced in human liver cancer cells, thecombination of Sorafenib with the inhibitor BIRB-796 shows a markedincrease in the efficacy over Sorafenib alone, and the combination of 12μM BIRB-796 with 2 μM Sorafenib results in a significant decrease inviable cancer cells compared to the reduction of cancer cells bySorafenib alone.

FIG. 10 shows crystal violet staining of cultivated human liver cancercells which are cultivated and treated for 7 days with 2 μM Sorafenib,12 μM BIRB-796, or a combination of 2 μM Sorafenib+12 μM BIRB-796 (upperrow), controls without kinase inhibitor are shown in the lower row(carrier). This view of the culture plates makes it evident that thecombination of Sorafenib with an inhibitor for Mapk14, represented byBIRB-796, increases the efficacy of Sorafenib.

The dead cell count of FIG. 11, using trypane blue staining of thecultivated human liver cancer cells (Hep3B) shows that the increase indead cells in the presence of an inhibitor (left hand columns) was moreprominent for the combination of Sorafenib with BIRB-796 over Sorafenibor BIRB-796 alone, and over the control (carrier, right hand columns)without a kinase inhibitor.

Similar to the assay for BIRB-796, SB 202190 was tested in an in vitroassay of the murine liver cancer cells (Nras Arf−/−) and the human livercancer cell line (Hep3B).

FIG. 12 shows the results of the viable cell count after two days oftreatment of the murine cancer cells and FIG. 13 shows the viable cellcount after 7 days of treatment of the cultivated human liver cancercell line (Hep3B) with the combinations of 2 μM Sorafenib, 2 μMSB202190, 12 μM SB202190, a combination of 2 μM Sorafenib+2 μM SB202190,or a combination of 2 μM Sorafenib+12 μM SB202190, respectively, in theleft columns, whereas samples without pharmaceutical active agent(carrier) are shown as right hand columns.

These results confirm that the activity of Sorafenib against livercancer cells can be reproduced in vitro with cultivated murine and humancancer cell lines respectively. Further, in murine cancer cells, andeven more pronounced in human cancer cells, the combination of Sorafenibwith the inhibitor SB202190 shows a marked increase in the efficacy overSorafenib alone, and the combination of 12 μM SB202190 with 2 μMSorafenib results in a significant decrease in viable cancer cellscompared to the reduction of cancer cells by Sorafenib alone.

FIG. 14 shows the crystal violet staining of cultivated human livercancer cells which are cultivated and treated for 7 days with 2 μMSorafenib, 12 μM SB202190, or a combination of 2 μM Sorafenib+12 μMSB202190 (upper row), controls without kinase inhibitor are shown in thelower row (carrier). This view of the culture plates makes it evidentthat the combination of Sorafenib with an inhibitor for Mapk14,represented by SB202190, increases the efficacy of Sorafenib.

The dead cell count shown in FIG. 15 (left hand columns indicate countsin presence of inhibitor, right hand columns are control with carrieronly) supports the finding that the combination of the Mapk14 inhibitorSB202190 with Sorafenib is significantly more effective than Sorafenibor SB202190 alone.

FIG. 16 shows the results of the viable cell counts after two days oftreatment of the murine cancer cells, and FIG. 17 shows the viable cellcount after 7 days of treatment of the cultivated human liver cancercell line (Hep3B) with the combinations of 10 μM or 2 μM Sorafenib, 52μM or 22 μM SX 011, and a combination of 10 or 2 μM Sorafenib+52 or 22μM SX 011 respectively, in the left hand columns, whereas sampleswithout pharmaceutical active agent (carrier) are shown as right handcolumns.

These results confirm that the activity of Sorafenib against livercancer cells can be reproduced in vitro with cultivated murine and humanliver cancer cells, respectively. Further, in murine cancer cells, andeven more pronounced in human cancer cells, the combination of Sorafenibwith the inhibitor SX 011 shows a marked increase in the efficacy overSorafenib alone, and the combination of SX 011 with Sorafenib results ina significant decrease in viable cancer cells compared to the reductionof cancer cells by Sorafenib alone.

1. Pharmaceutical composition comprising Sorafenib and an inhibitor ofthe activity of Mapk14 for use as a medicament in the treatment orprevention of liver cancer.
 2. Pharmaceutical composition according toclaim 1, wherein the inhibitor of the activity of Mapk14 is an siRNAcontaining an oligonucleotide having a nucleotide sequence hybridizingunder physiological conditions to the mRNA encoding Mapk14 of SEQ ID NO:1365, SEQ ID NO: 1366, SEQ ID NO: 1367 or SEQ ID NO:
 1368. 3.Pharmaceutical composition according to claim 2, wherein theoligonucleotide is selected from the group comprising SEQ ID NO: 1 toSEQ ID NO:
 1364. 4. Pharmaceutical composition according to claim 2,wherein the oligonucleotide comprises first section in a nucleic acidconstruct which contains a second section which is reverse complementaryto the first section.
 5. Pharmaceutical composition according to claim2, wherein the oligonucleotide is arranged under the control of apromoter in an expression cassette.
 6. Pharmaceutical compositionaccording to claim 2, wherein the oligonucleotide is contained in aliposome formulation and/or in a viral vector which is packaged in aviral particle or in a virus-like particle.
 7. Pharmaceuticalcomposition according to claim 1, wherein the inhibitor has an IC50 forMapk14 of at maximum 30 nM.
 8. Pharmaceutical composition according toclaim 7, wherein the inhibitor is selected from the group consisting ofthe compounds of table 1 in the specification.
 9. Pharmaceuticalcomposition according to claim 1, wherein Sorafenib is contained in aformulation for administration separate from a formulation containingthe inhibitor of the activity of Mapk14.
 10. Pharmaceutical compositionaccording to claim 1, wherein Sorafenib and the inhibitor of theactivity of Mapk14 are contained in one composition.
 11. Pharmaceuticalcombination for the treatment of or prevention of liver cancercomprising: a pharmaceutical formulation comprising an inhibitor of theactivity of Mapk14 and a separate pharmaceutical formulation comprisingSorafenib.
 12. Pharmaceutical combination according to claim 11, whereinthe pharmaceutical formulation comprising an inhibitor of the activityof Mapk14 is for administration separate from the administration of thepharmaceutical formulation comprising Sorafenib for the treatment orprevention of liver cancer for administration to a patient. 13.Pharmaceutical combination according to claim 11, wherein the inhibitorof the activity of Mapk14 is an siRNA containing an oligonucleotidehaving a nucleotide sequence hybridizing under physiological conditionsto the mRNA encoding Mapk14 of SEQ ID NO: 1365, SEQ ID NO: 1366, SEQ IDNO: 1367 or SEQ ID NO:
 1368. 14. Pharmaceutical combination according toclaim 11, wherein the inhibitor is selected from the group consisting ofthe compounds of table 1 in the specification.
 15. Method for treatmentor prevention of liver cancer comprising administration of an inhibitorof the activity of Mapk14 in combination with Sorafenib.
 16. Methodaccording to claim 15, wherein the inhibitor of the activity of Mapk14is an siRNA containing an oligonucleotide having a nucleotide sequencehybridizing under physiological conditions to the mRNA encoding Mapk14,the siRNA containing at least one of SEQ ID NO: 1 to SEQ ID NO: 1364, oran inhibitor which is a compound selected from the group consisting ofthe compounds of table 1 in the specification.